The dominant techniques for brain-wide functional imaging in humans and opaque mammals make use of he- modynamic contrast that results from coupling between neural activity and changes in blood flow and can be detected by noninvasive imaging methods including functional magnetic resonance imaging (fMRI), functional photoacoustic tomography (fPAT), functional ultrasound imaging (fUS), and others. Although hemodynamic functional imaging in both humans and animals has been used to make some of the most important discover- ies in neuroscience, the techniques are limited by their lack of specificity to mechanistically-distinct compo- nents of brain activity. Our group has helped lead efforts to bypass limitations of hemodynamic functional imag- ing by developing molecular probes that report physiologically-specific hallmarks of neural activity via noninva- sive imaging. Here we propose the complementary and hitherto unprecedented strategy of working ?within the system? to improve the specificity intrinsic hemodynamic readouts themselves. Specifically, we propose to ge- netically enhance a signaling pathway that directly couples the activity of targeted neurons or glia to blood ves- sels, thus introducing artificial hemodynamic functional signals that can be attributed to distinct cell type- or circuit-specific cellular processes in the brain. This approach will harness the amplification afforded by hemo- dynamic imaging while bypassing numerous complexities of endogenous neurovascular mechanisms, in effect hijacking hemodynamic signals to report on circuit- or cell type-specific activity. Compared with efforts based on more conventional imaging methods, the engineered hemodynamic imaging approach will offer key ad- vantages: (1) capability for selective imaging of genetically-targeted brain circuits and cell types, (2) compatibil- ity with a variety of genetic tools, in particular including viral transduction and tracing vectors, (3) applicability to brain-wide deep tissue imaging in multiple species, (4) compatibility with many established modalities for func- tional neuroimaging, (5) potential sensitivity to relatively low activity levels and sparse neuronal populations. Our work on this novel strategy will be organized into two Aims: In Aim 1 we will engineer a new family of genetically encodable neural activity reporters that will underlie the new engineered hemodynamic imaging approach. The reporters will be derived from naturally occurring nitric oxide synthase enzymes and are re- ferred to as NOSTICs. In Aim 2, we will deliver NOSTIC genes to rat brains using viral vectors, and perform an extensive series of imaging and histological investigations to validate and optimize our new imaging in vivo. We anticipate that completion of these Aims will introduce a potent new approach for multimodal imaging- based investigations of circuitry and cell type-specific contributions to neural function in diverse species and behavioral contexts.